Vijay Ramani,

My contributions to the scientific community thus far have been in inventing high-throughput sequencing-based assays capable of furthering our basic understanding of gene regulation. The first method I developed was RNA Proximity Ligation (2015), an unbiased, sequencing-based approach for resolving RNA secondary structure, at the scale of whole-transcriptomes. RNA Proximity Ligation adapted the concept of proximity ligation (used previously to measure protein-protein, protein-DNA, and DNA-DNA interactions) to study RNA secondary structure. Unlike previous high-throughput approaches for resolving RNA structure (e.g. SHAPE-seq, DMS-seq), which measured the relative likelihood that RNA bases were structured or unstructured, RNA Proximity Ligation revealed which specific pairs of regions interacted within intramolecular RNA structures. My ensuing projects focused on developing massively parallel single-cell assays to better understand transcriptional regulation. The methods that I developed employed the concept of combinatorial cellular indexing, which unites traditional sequencing-based gene regulatory assays with in situ DNA barcoding of single cellular nucleic acid content, to yield high-throughput, single-cell biochemical assays that do not require isolation of single cells. I adapted this paradigm to chromosome conformation capture (i.e. Hi-C), a single cell method we have termed sciHi-C (2017). SciHi-C fundamentally advanced measurement of single-cell chromosomal conformation; in our proof-of-concept publication, we presented over 10,000 single-cell conformational maps, two orders of magnitude more than the previously published example of single-cell Hi-C. Importantly, our single-cell sequencing approach can be generalized to any sequencing assay where DNA barcodes can be introduced in situ; I have also been involved in extending combinatorial cellular indexing to additional assays relevant to gene regulation, including RNA-seq (2017) and the simultaneous measurement of RNA and chromatin accessibility (2018).

My work at the forefront of genomic technology has uniquely positioned me to address a fundamental systems biological question—how tolerant are cells to perturbation of steady state abundances of key genes? To what extent do up- and downregulation of essential genes affect the broad spectrum of molecular “phenotypes” (e.g. replication timing, chromosome conformation, transcription, translation), and ultimately, cellular fitness? As a PI, I will remain at the forefront of developing novel techniques to assay gene regulatory phenomena. I will also apply these bulk- and single-cell genomic techniques to couple the regulatory circuits bridging cellular metabolism, nuclear chromatin architecture, and transcriptional output.